Precursors of silk-like materials, silk-like materials and processes for producing both

ABSTRACT

A silk-like material precursor, a method of producing such a precursor and a silk-like material produced from such a precursor are provided.  
     The silk-like material precursor is a copolymer represented by the general formula -[(GA 1 ) j -((GA 2 ) k -G-Y-(GA 3 ) l ) m ] n -. In the module (GA 1 ) j , the precursor is organized in a repeated β-turn type II structure having intra-molecular hydrogen bonds formed successively along its molecular axis. In this formula, G represents glycine and A 1  represents alanine, but it is also possible to replace every third A 1  with serine. A 2  and A 3  both represent alanine, but these residues may be partly replaced with valine. Y represents an amino acid containing an asymmetric carbon atom giving water solubility to the precursor. Finally, j is an integer of at least 6, k and l are both integers of 0 to 5, m is an integer of 1 to 7 and n is an integer of at least 10.

TECHNICAL FIELD

[0001] The present invention relates to precursors of silk-likematerial, a silk-like material produced therefrom, and methods ofproducing the same.

BACKGROUND OF THE INVENTION

[0002] Domestic silkworms (Bombyx mori silkworms) extrude liquid silk ina pre-fibrillation state and produce silk fibers with high strength andhigh elasticity within a very short time of spinning at roomtemperature. It is well known that fabric woven from such silk fibershas widely been praised for luxury clothing production. In addition,this kind of textile fabric is ecologically friendly since silk fibersare biodegradable. Therefore, in the past decades, natural silk hasbecome the subject of extensive studies as model systems. Although theprimary structure of Bombyx mori silk and the secondary structure ofsilk fibers (Silk II) have already been determined, the structure ofsilk before spinning (Silk I) and the conformational transition involvedhave not been clarified yet. Moreover, many attempts to produce suchsilk-like fibers with high strength and elasticity have been performedbut none of them really succeeded.

[0003] The primary structure of the silk fibroin produced by domesticsilkworms has been reported by Zhou et al. (Zhou et al., Nucleic AcidsResearch, 28, 2413-2419 (2000)). According to this report, thecrystalline repeated motifs (Gly-Ala-Gly-Ala-Gly-Ser)n and thesemicrystalline motifs containing Tyr and/or Val are partly included ina chain of Gly-Ala linkage. Furthermore, both the ordered and thedisordered domains are repeated in cycles. Besides, the structure ofsilk films (liquid silk extracted from the posterior silk glands andgently dried) has been studied on the atomic level by X-ray diffraction.In this case, the structure reported is namely Silk I. Synthesis andstructural analysis of several model compounds of silk have also beenperformed. However, the Silk I structure still remain poorlycharacterized. Therefore, the precise mechanism of formation of silkwormsilk fibers cannot be determined. Without this fundamental knowledge, itis undoubtedly difficult to produce silk-like fibers artificially.

[0004] Previous models of the Silk I structure include the well known“Crankshaft Model” proposed by Lotz et al. and the “Out-of-RegisterModel” proposed by Fossey et al. The former model is based on X-ray dataanalysis, electron-beam analysis and conformational energy calculations.The latter model is only based on elaborate conformational energycalculations. However, these two models cannot explain all the X-raydata. Moreover, the conformational transition from Silk I to Silk IIstructures requires the cleavage of inter-molecular hydrogen bonds andfurther, the formation of other hydrogen bonds. These requirements arenot fulfilled either for the “Crankshaft” or the “Out-of-Register Model”models since in these cases, all the hydrogen bonds are inter-molecular.

[0005] The present researcher has performed intensive studies on Silk Istructure by using new analytical methods based on solid-state nuclearmagnetic resonance (NMR). This researcher has also succeeded indetermining the Silk I structure precisely and the following inventionhas been achieved.

[0006] The first object of the invention is to provide the precursor ofsilk-like material, that is, the liquid silk.

[0007] The second object of the invention is to provide the method forproducing the precursor of silk-like material.

[0008] The third object of the invention is to provide silk-likematerials that can be synthesized from the precursor.

[0009] The fourth object of the invention is to provide a method ofproducing silk-like materials including silk.

DISCLOSURE OF THE INVENTION

[0010] The silk-like material precursor is designed as a copolymer(precursor) represented by the general formula-[(GA¹)_(j)-((GA²)_(k)-G-Y-(GA³)_(l))_(m)]_(n). The precursor isorganized in repeated β-turn type II structure wherein intra-molecularand inter-molecular hydrogen bonds are formed alternatively along themolecular axis. The whole invention proposes a method of producing sucha precursor, a liquid silk from this precursor and then silk-like fibersspun out from the silk solution.

BRIEF DESCRIPTION OF THE PICTURES

[0011] In FIG. 1 is depicted the Silk I structure of the silk-likeprecursor. Each broken line represents intra-molecular hydrogen bonds.The pictures presented in (a), (b) and (c) of FIG. 2 showinter-molecular hydrogen bonds formed by the silk-like materialprecursor having the Silk I structure. Additionally, the broken lines inthe FIG. 2b represent intra-molecular hydrogen bonds and those in theFIGS. 2a and (c) stand for inter-molecular hydrogen bonds.

BEST MODE FOR CARRYING OUT THE INVENTION

[0012] The formula -[(GA¹)_(j)-((GA²)_(k)-G-Y-(GA³)_(l))_(m)]_(n)-representing the silk-like material precursor is not necessarily appliedto the chain ends. In the above formula, G represents glycine and Yrepresents an amino acid having an asymmetric carbon atom, allowingwater solubility to the precursor. Examples of such an amino acidinclude L-glutamic acid, L-lysine and L-tyrosine, the latter beingparticularly suitable. Furthermore, “L-” stands for a levo-rotatoryoptical isomer. A¹ represents alanine but it is also possible to replaceevery third alanine with serine. A² and A³ both represent alanine, butthey may be partly replaced with valines.

[0013] The formula -[(GA¹)_(j)-((GA²)_(k)-G-Y-(GA³)_(l))_(m)]_(n)- isrepresented in a block of copolymer form. The present precursor is notconfined to those having a block of copolymer form though. For thesubscript letters, j is an integer of at least 6, k and l are bothintegers of 0 to 5, m is an integer of 1 to 7 and n is an integer of atleast 10. Additionally, Y may include different kinds of amino acids,but it is especially advantageous that Y represents L-tyrosine alone.The water solubility of the precursor varies depending on the content ofY and it is appropriate that the content of Y is 20 mole % or below.Although n is an integer of at least 10 as defined above, it isdesirable for the spinning point of view capability that n is a valueenabling the copolymer to have a molecular weight of at least 30,000.

[0014] The term “repeated β-turn type II structure” used in thedescription of the invention represents a structure in which, asillustrated in FIG. 1, the amino acid residues are arranged in repeatedturns having a helical fashion. In contrast to the Silk II structure andto the traditional models, such repeated β-turn type II structureenables the precursor, represented by the formula mentioned above, tosequentially form intra-molecular hydrogen bonds along its molecularaxis. These intra-molecular hydrogen bonds are represented by brokenlines in FIG. 1 and FIG. 2(b). A method of producing the presentsilk-like material precursor is described in details below.

[0015] Before to produce the precursor, the amounts of amino acids to beused together with glycine are first specified. Furthermore, the amountof Y is determined depending on the desired water solubility. Accordingto the total amino acids composition, it is appropriate that theproportion of Y is 20 mole % or below. When the proportion of Y isincreased beyond 20 mole %, the stability of the precursor decreases andprevents the fibrillation to occur. Moreover, it is required that Y isintroduced in the main chain. The hardness of the copolymer can also becontrolled by introducing a serine residue in every third position of asequence of alanines represented by A¹. The appropriate proportion ofserine has to be considered as well.

[0016] The precursor can be synthesized using a solid-phase peptidesynthesis method or a genetic engineering method using bacteria(Escherichia coli). When the latter method is adopted to produce acopolymer having a high molecular weight, the general formula of theprecursor is determined first. Then, the sequence of oligonucleotidesinto which the desired sequence is encoded is synthesized chemically. Byusing these oligonucleotides, the repetitive unit of the general formulais produced. If it is necessary, this sequence is incorporated into acloning vector and then transferred into Escherichia coli. The last stepwill allow the polymerization of the precursor to an appropriate extend.In the present invention, it is advantageous to adopt the geneticengineering method characterized by the aforementioned steps. Ananalogous method is also enclosed in the Prince J, T et al. work(Biochemistry 34, 10879 (1995)).

[0017]FIG. 1 is a picture presenting the Silk I structure of a copolymerof glycine and alanine, wherein each intra-molecular hydrogen bond isformed along the molecular axis. The pictures in FIG. 2 demonstrate aregular inter-molecular structure. More specifically, the pictures (a),(b) and (c) are c-axis projection, a-axis projection and b-axisprojection, respectively. In addition to the well-ordered structurerepresented by the Silk I structure, the precursor may contain amorphousregions as well.

[0018] In a molecule adopting the Silk I structure, as shown in FIG. 1and FIG. 2(b), three amino acid residues form an intra-molecularhydrogen bond between the amino group located at one end (e.g.,Ala(i+3)) and the oxygen atom of the carbonyl group positioned at theother end (e.g., Gly(i)). By forming such intra-molecular hydrogen bond,the present copolymer can assume a cyclic structure referred to the“β-turn type II structure”. As shown in FIG. 1, the two peptide groupsparticipating in the intra-molecular hydrogen bonding of the β-turn typeII structure are also shared by other chain with β-turn type IIstructure.

[0019] As shown in FIG. 2(a) and FIG. 2(c), when the molecules organizedin the so-called Silk I structure are in the solid state, the adjacentmolecules along the a-axis direction are bounded by inter-molecularhydrogen interactions. As depicted in FIG. 1, each inter-molecularhydrogen bond is formed between amino acid residues whose are notparticipating to intra-molecular hydrogen bonding. More specifically,the Silk I solid-state structure is composed of amino acid residuesparticipating in intra-molecular hydrogen bonding and amino acidresidues contributing to inter-molecular hydrogen bonding, whichalternate with each other.

[0020] The formation of intra-molecular hydrogen bonds in Silk Istructure can be confirmed by determining the distance between thecarbon and the nitrogen atoms participating in the intra-molecularbonding by using a recent solid-state NMR method named REDOR. Thistechnique is very useful to determine accurately the distance betweentwo atoms labeled (e.g. ¹³C and ¹⁵N).

[0021] Overall, as described below, the repeated β-turn type II modelgives a reasonable explanation for the mechanism of silk fiber formationfrom liquid silk extruded by domestic silkworms.

[0022] The present precursor of Silk I structure has a high thermalstability in the solid state. However, by stretching along the fiberaxis direction, the Silk I structure changes easily to Silk IIstructure. Such a transition can be understood looking at theintra-molecular hydrogen bonds whose are approximately parallel to themolecular axis. Moreover, this conformational transition can also beunderstood in terms of close packing of the individual chains, as shownin FIG. 1 and FIG. 2(b). As we can observe in the Silk I structure, theintra-molecular hydrogen bonds are almost parallel to the molecular axisand so these bonds are easily broken upon stretching along this axis.Then, the inter-molecular hydrogen bonds are newly formedperpendicularly to the molecular axis. The individual sets of aminoacids participating in the intra-molecular hydrogen bonds of eachmolecule stands in proximity and are facing the residues involved inintra-molecular hydrogen bonds present in adjacent molecules.Consequently, the formation of inter-molecular hydrogen bonds occurssimultaneously with the cleavage of intra-molecular hydrogen bonds inthe Silk I structure. This passage can be referred as the “transition ofintra-molecular hydrogen bonds”.

[0023] In the present invention, a copolymer is synthesized inEscherichia coli, purified and dissolved in LiBr aqueous solution. TheLiBr is removed from the solution by dialysis and the water is thengently evaporated. Thereby, the precursor is obtained. In the process ofremoving water, a silk film is usually obtained. At this step, when theinitial aqueous solution has a low silk concentration, the precursor canbe amorphous. It is also possible to let the present precursor adopt theSilk I structure by having an initial aqueous solution with a highersilk concentration. The Silk I structure obtained can also be convertedinto the Silk II structure by stretching the film or treat it withformic acid. However, in order to produce silk fibers, it is required tospin out the silk from the aqueous solution before the film formationoccurs. It is also possible to produce silk fibers by dissolving theprecursor obtained as a film, e.g., in hexafluoroisopropanol (HFI) orso, and then spin out the fibers from the resulting solution. Thespinning conditions may be determined appropriately according to thephysical properties of the precursor obtained. Furthermore, by selectingthe orifice opening of a spinning nozzle, it is possible to producesilk-like fibers having various diameters. Basically, for low productioncosts and eco-friendliness, it is preferable to avoid the use of asolvent such as hexafluoroisopropanol. It is certainly more appropriateto spin out the silk-like precursor from an aqueous solution. However,it is also possible to design the precursor such that it can bedissolved in organic solvents such as alcohol or ether.

[0024] In general, several functions can be added to the silk-likematerial, prepared either as a film or as a fiber, by modifying thecomposition of the precursor. Moreover, the precursor is a protein andis degradable upon the action of microbes. Besides, it has the uniqueproperties of natural silk and its method of production is ecological.

[0025] The present invention will be illustrated in more details with afew examples. It is important to stress the fact that these examplesshould not be considered as limiting for the scope of the presentinvention.

EXAMPLES Example 1

[0026] Comparative examinations of conformational transitions of thetyrosine residues present in silk were made. The silk samples wereprepared from domestic silkworms and were isotopically labeled in ¹³Cfor the L-tyrosine residues. To do so, 50 mg of ¹³C-labeled L-tyrosinewas given to silkworm larvae in the fifth instar stage. The silk sampleswere then characterized by solid-state high-resolution ¹³C NMRexperiments. It was confirmed that the L-tyrosines took random coilconformations in both liquid silk and film samples formed by drying.However, these residues were rather organized in Silk II structures whenthe silk was spun out to form the cocoons. Silk fibers prepared bydissolving the silk films in hexafluoroisopropanol and spun out from theresulting solution were also studied by orientated-solid state NMRmeasurements. It was clear that L-tyrosine were involved in Silk IIstructures. In each molecular chain, these residues were also wellorientated. In addition, it was confirmed by solid-state NMRmeasurements that both alanine and glycine residues, whose are the mainconstituents of silk, took the Silk I structure either in the liquidsilk and or in the dried film samples but they were rather involved inthe Silk II structure in the silk fibers.

Example 2

[0027] The following peptides I, II, III and IV, whose arerepresentative chain segments in the repeated domains of Bombyx morisilk, were synthesized using solid-phase methods. The solubility inwater was also examined. The model compound I was insoluble in water butthe peptides II, III and IV were highly soluble. By these examinations,it was therefore verified that tyrosine residues are essential forgiving solubility to silk in water.

[0028] Synthesized Compounds: G(AG)₂SG(AG)₂ I G(AG)₂YG(AG)₂ IIGAGVGYG(AG)₂ III TGFDSESSWAYEYGSYGGNAVYPGFGSSGT IV

[0029] Herein, G stands for glycine, A for alanine, Y for tyrosine, Vfor valine, T for threonine, F for phenylalanine, D for aspartic acid, Efor glutamic acid, W for tryptophan, N for asparagine and P for proline.All these amino acids are L-amino acids excepted glycine.

Example 3

[0030] The following model compounds of silk were synthesized usingsolid-phase methods. G(AG)₂YG(GA)₂ II (AG)₆YG(AG)₅ V (AG)₇YG(AG)₇ VI(AG)₁₅ VII

[0031] Herein, A stands for L-alanine, G for glycine and Y forL-tyrosine.

[0032] As mentioned in the Example 2, the sample II was highly solublein water. The Sample V was studied as a gel with solid-statehigh-resolution ¹³C NMR. In this case, the Silk I structure was adopted.The gel was prepared by dissolving 300 mg of Sample V in 7 ml of a 9MLiBr aqueous solution. In order to remove the LiBr, the solution wasthen dialyzed for 3 days and the sample had a gel appearance in themembrane used for the dialysis. Through the same procedure mentionedabove, a gelatin texture was also observed for the Sample VI. However,both structures Silk I and Silk II were present in this sample. Finally,for the sample VII, a precipitate was formed in the membrane fordialysis. This precipitate was found to adopt the Silk I structure.

Example 4

[0033] The following model compounds of silk were synthesized usingsolid-phase methods. (AG)₃YG(AG)₃YG(AG)₃YG(AG)₃ VIII(AG)₃YG(AG)₂VGYG(AG)₃YG(AG)₃ IX (AGAGYG)₅ X

[0034] An amount of 300 mg of the samples VIII, IX and X was dissolvedin 7 ml of a 9M LiBr aqueous solution. The solution was then dialyzedfor 3 days to remove the LiBr. During the dialysis process, all peptidestook a gel-like form in the membrane for dialysis. As suited, theresulting materials presented high water contents. During the dialysisprocess, the gel-like form was clearly different from the one observedfor the sample VII ((AG)₁₅). Thus the tyrosine residues introduced ineach copolymer had proved to give a hydrophilic character to thecopolymer.

Example 5

[0035] In order to prepare an aqueous solution of silk fibroin, 1 g ofdegummed fibers (without sericin) were dissolved in 50 ml of a 9M LiBraqueous solution and kept at 40° C. for 1.5 hours. The insolublecomponents were filtered from the solution. An aqueous solution of 9MLiBr with a silk concentration of 0.5% was obtained. The solution wasthen transferred into a membrane and dialyzed against distilled waterfor 4 days. The aqueous solution with a silk concentration lower than0.5% was prepared as well. The silk solution was spread on No.2 squarePetri dishes and dried moderately at room temperature. An amount of 0.15g per sheet was obtained from the resulting amorphous films. These filmswere soluble in HFIP at room temperature (15° C. to 30° C.), and it tookabout 3.5 hours for a complete dissolution of each film in the solvent.The fibers spun out from the HFIP solution were drawn and theirstructure was characterized. As a result, it was confirmed that thedrawn fibers took the Silk II structure.

Example 6

[0036] The following model compounds of silk were synthesized usingsolid-phase methods. (AG)₆A[1-¹³C]G [1-¹³C]AG(AG)₇ XI(AG)₇[1-¹³C]A[1-¹³C]G(AG)₇ XII (AG)₆A[1-¹³C]GA[1-¹³C]G(AG)₇ XIII(AG)₆A[1-¹³C]GA[¹⁵N]G(AG)₇ XIV (AG)₇[1-¹³C]AG[¹⁵N]AG(AG)6₇ XV

[0037] An amount of 300 mg of each compound was dissolved in 7 ml of a9M LiBr aqueous solution and dialyzed for 3 days leading to theformation of a precipitate in the dialysis membrane. IR spectraindicated that the precipitate adopts Silk I structure. Two-dimensional¹³C solid-state NMR spin diffusion experiments were performed on themodel compounds XI, XII and XIII. REDOR NMR measurements were also usedto characterize the samples XIV and XV. The symbols [1-¹³C]G and[1-¹³C]A represent glycine and alanine residues in which the carbon ofthe carbonyl group is labeled with ¹³C, respectively. The symbols [¹⁵N]Gand [¹⁵N]A represent glycine and alanine residues labeled with ¹⁵N,respectively.

[0038] Based on spectral analysis, the torsion angles (φ,φ) of thealanine residues included in the model compound XI were determined andare the following: −60°±5° and 130°±5°, respectively. The torsion angles(φ,φ) of the glycine residues included in the model compounds XII andXIII were also determined and are the following: 70+±5° and 30°±5°,respectively. By REDOR measurements, it has also been found that thetorsion angles (φ,φ) are the same for the alanine and glycine reissuedincorporated in the compounds XIV and XV.

[0039] Finally, when the torsion angles of a copolymer composed byalternating alanine and glycine residues were adjusted to the valuesdetermined above, the copolymer adopted the structure shown in FIG. 1(Silk I).

Example 7

[0040] The following model compounds of silk were synthesized usingsolid-phase methods. (AG)₁₅ VII (AG)₆A[1-¹³C]GAG [¹⁵N]AG(AG)₆ XVI

[0041] As presented in the Example 1, these samples were prepared suchthat the Silk I structure is formed. By REDOR measurements of the sampleXVI, the distance between the [1-¹³C]G₁₄ residue and the [¹⁵N]A₁₇residue was determined to be 4.0±0.1 Å. Such a distance was alsomeasured for an equal mixture of samples XVI and VII. Therefore, itseems that there is no inter-molecular influence on the determination ofatomic distances. Moreover, the distance 4.0±0.1 Å is in good agreementwith the distance between the corresponding atoms in the structuredepicted in FIG. 1. This fact proves clearly that Silk I structure formsintra-molecular hydrogen bonding.

EXAMPLE 8

[0042] Degummed silk fibroin fibers (1 g) were added to a 9M LiBraqueous solution (50 ml) and kept at 40° C. for 1.5 hours. The resultingsolution was filtered in order to remove the insoluble components. Anaqueous solution of 9M LiBr with a silk concentration of 0.5% wasobtained and transferred into a membrane and dialyzed against distilledwater for 4 days. The aqueous solution with a silk concentration lowerthan 0.5% was prepared as well. The pH of the silk solution (containing4 g of silk) was adjusted to 7.8 by addition of sodium phosphate. Then,several ml of an aqueous solution containing chymotrypsin was added tothe solution and followed by a 24-hours incubation at 40° C. Theprecipitate was centrifuged at 10,000 r.p.m. and the enzymatic reactionwas ended by the addition of 0.03 N hydrochloric acid. The precipitatewas then washed and a CP fraction of about 55% of the original silkfibroin was obtained. The sequence of the CP fraction has beenpreviously characterized and is mainly composed by repetitive motifs ofAGAGSG (Strydom et al., Biochem. Biophys. Res. Commun., 3, 932 (1977)).

[0043] The CP fraction we obtained was dissolved in an aqueous solutionof 9M LiBr and dialyzed. By IR measurements, we observed that the samplewas adopting the so-called Silk I structure. ¹³C solid-statehigh-resolution NMR spectra of this CP fraction and the model compoundVII ((AG)₁₅) were acquired. As a result, it was found that the peaksposition and the line shapes arising from the alanine and glycineresidues were the same for these two samples. Thus, it can be concludedthat the present alanine-glycine copolymer is a representative model ofthe crystalline domain (AGAGSG)n of Bombyx mori silk. By treating thesamples in formic acid, the conformational transition from Silk I toSilk II occurs. In this case, the peaks position arising from thealanine and glycine residues is also in agreement between the twosamples.

INDUSTRIAL APPLICABILITY

[0044] According to our invention, Silk-like materials may havedifferent functions by modifying some constituents in the copolymer.Moreover, the precursor presents the unique properties of natural silkwhen spun into fibers. Since the copolymer is a protein, it isbiodegradable and therefore, our method of producing this silk-likematerial is ecological.

1 19 1 11 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 1 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 1 5 10 211 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 2 Gly Ala Gly Ala Gly Tyr Gly Ala Gly Ala Gly 1 5 10 3 11 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide3 Gly Ala Gly Val Gly Tyr Gly Ala Gly Ala Gly 1 5 10 4 30 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 4 Thr GlyPhe Asp Ser Glu Ser Ser Trp Ala Tyr Glu Tyr Gly Ser Tyr 1 5 10 15 GlyGly Asn Ala Val Tyr Pro Gly Phe Gly Ser Ser Gly Thr 20 25 30 5 24 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide5 Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Tyr Gly Ala Gly 1 5 1015 Ala Gly Ala Gly Ala Gly Ala Gly 20 6 30 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 6 Ala Gly Ala GlyAla Gly Ala Gly Ala Gly Ala Gly Ala Gly Tyr Gly 1 5 10 15 Ala Gly AlaGly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly 20 25 30 7 30 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 7 Ala GlyAla Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly 1 5 10 15 AlaGly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly 20 25 30 8 30 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide8 Ala Gly Ala Gly Ala Gly Tyr Gly Ala Gly Ala Gly Ala Gly Tyr Gly 1 5 1015 Ala Gly Ala Gly Ala Gly Tyr Gly Ala Gly Ala Gly Ala Gly 20 25 30 9 30PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 9 Ala Gly Ala Gly Ala Gly Tyr Gly Ala Gly Ala Gly Val Gly TyrGly 1 5 10 15 Ala Gly Ala Gly Ala Gly Tyr Gly Ala Gly Ala Gly Ala Gly 2025 30 10 30 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 10 Ala Gly Ala Gly Tyr Gly Ala Gly Ala Gly Tyr Gly AlaGly Ala Gly 1 5 10 15 Tyr Gly Ala Gly Ala Gly Tyr Gly Ala Gly Ala GlyTyr Gly 20 25 30 11 30 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 11 Ala Gly Ala Gly Ala Gly Ala Gly Ala GlyAla Gly Ala Gly Ala Gly 1 5 10 15 Ala Gly Ala Gly Ala Gly Ala Gly AlaGly Ala Gly Ala Gly 20 25 30 12 30 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 12 Ala Gly Ala Gly Ala Gly AlaGly Ala Gly Ala Gly Ala Gly Ala Gly 1 5 10 15 Ala Gly Ala Gly Ala GlyAla Gly Ala Gly Ala Gly Ala Gly 20 25 30 13 30 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 13 Ala Gly Ala GlyAla Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly 1 5 10 15 Ala Gly AlaGly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly 20 25 30 14 30 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide14 Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly 1 510 15 Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly 20 25 3015 30 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 15 Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly AlaGly Ala Gly 1 5 10 15 Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala GlyAla Gly 20 25 30 16 30 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 16 Ala Gly Ala Gly Ala Gly Ala Gly Ala GlyAla Gly Ala Gly Ala Gly 1 5 10 15 Ala Gly Ala Gly Ala Gly Ala Gly AlaGly Ala Gly Ala Gly 20 25 30 17 6 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 17 Ala Gly Ala Gly Ser Gly 1 5 186 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 18 Gly Ala Gly Ala Gly Ser 1 5 19 2380 PRT Artificial SequenceDescription of Artificial Sequence Formula sequence for silk-likematerial precursor 19 Gly Ala Gly Ala Gly Xaa Gly Ala Gly Ala Gly XaaGly Xaa Gly Xaa 1 5 10 15 Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa GlyXaa Gly Xaa Gly Xaa 20 25 30 Gly Xaa Gly Ala Gly Ala Gly Xaa Gly Ala GlyAla Gly Xaa Gly Xaa 35 40 45 Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa GlyXaa Gly Xaa Gly Xaa 50 55 60 Gly Xaa Gly Xaa Gly Ala Gly Ala Gly Xaa GlyAla Gly Ala Gly Xaa 65 70 75 80 Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly XaaGly Xaa Gly Xaa Gly Xaa 85 90 95 Gly Xaa Gly Xaa Gly Xaa Gly Ala Gly AlaGly Xaa Gly Ala Gly Ala 100 105 110 Gly Xaa Gly Xaa Gly Xaa Gly Xaa GlyXaa Gly Xaa Gly Xaa Gly Xaa 115 120 125 Gly Xaa Gly Xaa Gly Xaa Gly XaaGly Ala Gly Ala Gly Xaa Gly Ala 130 135 140 Gly Ala Gly Xaa Gly Xaa GlyXaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa 145 150 155 160 Gly Xaa Gly Xaa GlyXaa Gly Xaa Gly Xaa Gly Ala Gly Ala Gly Xaa 165 170 175 Gly Ala Gly AlaGly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa 180 185 190 Gly Xaa GlyXaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Ala Gly Ala 195 200 205 Gly XaaGly Ala Gly Ala Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa 210 215 220 GlyXaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Ala 225 230 235240 Gly Ala Gly Xaa Gly Ala Gly Ala Gly Xaa Gly Xaa Gly Xaa Gly Xaa 245250 255 Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa260 265 270 Gly Ala Gly Ala Gly Xaa Gly Ala Gly Ala Gly Xaa Gly Xaa GlyXaa 275 280 285 Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly XaaGly Xaa 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1. The silk-like material precursor characterized as a copolymerrepresented by the general formula-[(GA¹)_(j)-((GA²)_(k)-G-Y-(GA³)_(l))_(m)]_(n)-, containing repeatedβ-turn type II structures in the module (GA¹)_(j) and havingintramolecular hydrogen bonds formed successively along its molecularaxis. In this formula, G represents glycine, A¹ represents alanine, butit is also possible to replace every third A¹ with serine, A² and A³both represent alanine, but these residues may be partly replaced withvaline. Y represents an amino acid with an asymmetric carbon atom givingwater solubility to the precursor. The j is an integer of at least 6, kand l are both integers of 0 to 5, m is an integer of 1 to 7 and n is aninteger of at least
 10. 2. In the precursors of silk-like material asdescribed in the claim 1, Y in the formula is L-tyrosine and this shouldbe contained in a proportion lower than 20 mole %.
 3. The silk-likematerial precursors as described in claims 1 or 2, wherein Y in theformula is L-tyrosine.
 4. A method for producing the silk-like materialprecursors with the following characteristics. The copolymers weredesigned according to the general formula defined in claim
 1. Then, atfirst, the oligonucleotides with the desired sequence is chemicallysynthesized and the repetition units in the formula are prepared usingthese oligonucleotides. Then it is incorporated into a cloning vector inorder to do the polymerization to an appropriate extent. Afterincorporating the repetition units or its polymers into an expressionvector, the vector is transferred into Escherichia coli in order toexpress the precursor.
 5. The silk-like materials which werecharacterized by spinning of the silk-like material precursors describedin any of claims 1 to 3 lead to a transition from the intra- andinter-molecular hydrogen bonding formations along the chain to allinter-molecular hydrogen bonding formations.
 6. The silk-like materialsas described in claim 5, wherein the spinning is performed under awater-containing condition.
 7. A method of producing a silk-likematerials with Silk II structure. This is characterized by spinning fromthe solution containing the silk-like material precursors as describedin claim 1, where the precursor with intra-molecular hydrogen bondschanges to the sample with intermolecular hydrogen bonds by spinning andthen drawing, if necessary.
 8. A method of producing silk-like materialsas described in claim 7, wherein the solution of the silk materials isprepared as follows. The dried films with amorphous or Silk I-structureare prepared from the aqueous solution of the silk-like materialprecursors and then dissolved in hexafluoroisopropanol at temperature of15° C. to 30° C.